One of the major unanswered questions in the regulation of erythropoiesis is the mechanism by which hypoxia triggers increased production of erythropoietin (Epo). In the fetus, the liver is the primary site of Epo synthesis whereas after birth production shifts to the kidney. We have shown that the human hepatoma cell line Hep3B produces high levels of Epo mRNA and protein following exposure to hypoxia or treatment with cobalt chloride, an agent previously shown to increase Epo production in vivo. Nuclear run-off and actinomycin chase experiments will be performed in order to ascertain whether the regulation of Epo mRNA is solely at the transcriptional level. The cis-acting regulatory elements of the Epo gene will be investigated in both transient and stable expression experiments using Hep3B and control cell lines transfected with non- coding regions of the Epo gene linked to a reporter gene (human growth hormone). The production of growth hormone following challenge with hypoxia or cobalt will be measured following transfection with a comprehensive set of constructions. Mapping of the DNAse sensitive sites of the EPO gene will provide independent information on the gene's regulatory elements. An additional major aim of this proposal is to better understand the biochemical nature of the oxygen sensor. Experiments have been designed to test the hypothesis that the sensor is a heme protein whose conformation depends on the metal atom within the porphyrin ring and whether it is bound to oxygen. The trans-acting factors that regulate the Epo gene will be investigated by comparison of nuclear extracts prepared from control cells versus those challenges with cobalt or hypoxia. Gel retardation and footprinting analyses will be employed to identify those regions of Epo DNA that display hypoxia- and cobalt-specific protein binding. These findings will be correlated with the results of the transection experiments described above. Stretches of DNA that appear to function as regulatory elements will be attached to Sepharose and used to affinity purify specific trans-acting DNA binding proteins. Special attention will focus on heme-containing proteins. The Epo response of the Hep3B cell line to hypoxia and cobalt is comparable to that observed in vivo. Therefore it is an ideal model system to gain a coherent understanding of the molecular circuit leading from the hypoxia signal to enhanced expression of the Epo gene.